High resolution respirometry of isolated mitochondria from adult Octopus maya (Class: Cephalopoda) systemic heart

Mitochondrial respirometry is key to understand how environmental factors model energetic cellular process. In the case of ectotherms, thermal tolerance has been hypothesized to be intimately linked with mitochondria capability to produce enough adenosine triphosphate (ATP) to respond to the energetic demands of animals in high temperatures. In a recent study made in Octopus maya was proposed the hypothesis postulating that high temperatures could restrain female reproduction due to the limited capacity of the animals’ heart to sustain oxygen flow to the body, affecting in this manner energy production in the rest of the organs, including the ovarium Meza-Buendia AK et al. (2021). Unfortunately, until now, no reports have shown temperature effects and other environmental variables on cephalopod mitochondria activity because of the lack of a method to evaluate mitochondrial respiratory parameters in those species’ groups. In this sense and for the first time, this study developed a method to obtain mitochondrial respirometry data of adult Octopus maya’s heart. This protocol illustrates a step-by-step procedure to get high yield and functional mitochondria of cephalopod heart and procedure for determining the corresponding respiratory parameters. The procedure described in this paper takes approximately 3 to 4 hours from isolation of intact mitochondria to measurement of mitochondrial oxygen consumption.

ABSTRACT Mitochondrial respirometry is key to understand how environmental factors model energetic cellular process. In the case of ectotherms, thermal tolerance has been hypothesized to be intimately linked with mitochondria capability to produce enough adenosine triphosphate (ATP) to respond to the energetic demands of animals in high temperatures. Recent studies made in Octopus maya proposed the hypothesis postulating that high temperatures could restrain female reproduction due to the limited capacity of the animals' heart to sustain oxygen flow to the body, affecting in this manner energy production in the rest of the organs, including the ovarium. Until now, no reports have shown temperature effects and other environmental variables on cephalopod mitochondria activity because of the lack of a method to evaluate mitochondrial respiratory parameters on those groups of species. In this sense and for the first time, this study developed a method to obtain mitochondrial respirometry data of adult Octopus maya's heart. This protocol illustrates a step-by-step procedure to get high yield and functional mitochondria of cephalopod heart and procedure for determining the corresponding respiratory parameters. The isolation procedures described here require two hours, demonstrating that confident and replicable results can be obtained with this method. 4 Cut the systemic heart into pieces with scissors and mince into smaller pieces with a scalpel, which should be done while the Petri dish is on ice.
5 Transfer the cut pieces of the organ to a homogenization tube with 2 ml of cold mitochondrial isolation buffer A.
NOTE: NOTE: Homogenization, as well as the following steps, must be carried out at 4 °C. 6 Homogenize the systemic heart using Potter-Elvehjem PTFE pestle and glass tube (Sigma-Aldrich P7859-1EA) homogenizer operated by a drill at 500 rpm. Three to four stocks are made to homogenize the previously minced tissue. Homogenization is done in a container with ice and the ice homogenization tube must not be removed.
CRITICAL STEP: CRITICAL STEP: The drill pistil must enter rotating to avoid forming bubbles and generating surface tension causing the isolated mitochondria to burst.

17
The following protocol is designed to be used in a commercially available HRR device, the Oxygraph™ O2k (Oroboros Instruments, Innsbruck, AT), which uses a polarographic oxygen sensor to detect oxygen (O₂) flux of ± 1 pmol O₂·s⁻¹·mL⁻¹. To adapt the protocol to other commercial equipment, please see the manufacturer's specifications. The equipment should be turned on before the mitochondrial isolation starts, so it reaches the selected experimental working temperature (the data shown in this document were determined at temperature of 24 °C). This section provides a SUIT protocol for the analysis of oxidative phosphorylation (OXPHOS) in Octopus maya systemic heart mitochondria, being a tool for understanding the mitochondrial respiratory control of this species. See Table 1, to consult the concentrations of the substrates and inhibitors used in this protocol. to obtain a final concentration between 300-500 μg ml -1 . This step is followed by a rapid and transient decrease in oxygen content of the chamber followed by a slower decrease caused by respiration of the mitochondria, commonly referred to as Respiratory State 1. CRITICAL STEP CRITICAL STEP : The corresponding respiratory substrates must be immediately added to avoid mitochondrial membrane potential depolarization. NOTE: NOTE: The addition of proline starts proline pathway (entry in electron transport system direct into Q-junction) and the glutamate-anaplerotic pathway (stimulates CIlinked respiration). Proline is oxidized to 1-delta pyrroline 5 carboxylate by proline dehydrogenase of the inner mitochondrial membrane reducing FAD to FADH2, where 1-delta pyrroline 5 carboxylate is converted to glutamate by 1 pyrroline 5 carboxylate dehydrogenase. Additionally, FADH2 is oxidized to stimulate quinone reduction, activating Q-junction.
24 Observe a faster rate of oxygen consumption because of basal activity of the respiratory chain to counteract proton leakage from the inner mitochondrial membrane, which represents Respiratory State 2' (S2' S2'). NOTE: NOTE: To correctly determine O₂ consumption rate in each Respiratory State, it is necessary to ensure that the steady-state is reached. NOTE NOTE: To avoid hypoxia in the chambers, they must be reoxygenated (by chamber opening) if O 2 concentration falls below 20 µM.